三丁基膦(TBP)和二硫苏糖醇(DTT)在秦川牛肌肉组织双向电泳中使用效果的差异及原因分析  

Comparison of Tributyl phosphine(TBP) and DL-Dithiothreitol(DTT)for Two-dimensional Gel Electrophoresis(2-DE) of Qinchuan Cattle(Bos taurus) Longissimus Muscle Tissue

作　　者：郝瑞杰[1] 赵宪林[1] 李安宁[1,2] 王嘉力[1] 赵春平[1,2] 王洪宝[1,2] 成功[1,2] 杨武才[1,2] 昝林森[1,2] 

机构地区：[1]西北农林科技大学动物科技学院,杨凌712100 [2]国家肉牛改良中心,杨凌712100

出　　处：《农业生物技术学报》2015年第4期546-553,共8页Journal of Agricultural Biotechnology

基　　金："十二五"国家863计划(No.2013AA102505;No.2011AA100307-02);国家自然科学基金(No.31272411);国家肉牛牦牛产业技术体系(CARS-38);国家科技支撑计划(No.2012BAD28B04-03);国家转基因动物育种专项(No.2013ZX08007-002);陕西省科技创新工程计划(No.2014KTZB02-02-01)

摘　　要：合适的还原剂对蛋白的完全溶解以及较好双向电泳结果的获得至关重要。三丁基膦(tributyl phosphine,TBP)通常被认为是比二硫苏糖醇(DL-Dithiothreitol,DTT)更为有效的还原剂。本实验首次从图谱质量和蛋白分布两个方面,详细对比了TBP和DTT在秦川牛(Bos taurus)肌肉组织全蛋白和肌质蛋白双向电泳中的使用效果。结果表明,和TBP相比无论是全蛋白还是肌质蛋白,使用DTT作为还原剂的图谱背景均更为清晰,分辨率更高,对高分子量蛋白以及低丰度蛋白的提取也更为有效。因此,DTT比TBP更适用于秦川牛肌肉组织双向电泳。本研究的结果和传统的观点并不一致,初步分析了该原因,可能是TBP较短的半衰期导致了其在双向电泳中较差的使用效果。该实验为双向电泳的样品制备以及牛肌肉组织蛋白质组学研究提供了技术资料。Appropriate reducing agent is very crucial for complete dissolution of protein and better results in two-dimensional gel electrophoresis （2-DE）. Tributyl phosphine （TBP） is considered to be a more effective reducing agent than DL-Dithiothreitol （DTT） in general. In this study, we detailly compared DTT with TBP in term of 2-DE map quality and protein spot distribution of total proteins and sarcoplasmic proteins from Qinchuan cattle （Bos taurus） longissimus muscle tissue for the first time. The result implied that the reducing agent of DTT produced a higher resolution 2-DE gels with a clearer background than TBP. For the total proteins, DTT extracted more proteins with molecular weight 〉20 kD and protein volume 〈 1 000 than TBP. For the sarcoplasmic proteins DTT promoted extraction of proteins with molecular weight〉 50 kD and protein volume〈5 000 compared with TBP. DTT was more appropriate to 2-DE of Qinchuan cattle longissimus muscle tissue than TBP and this result wasn＇t consistent with traditional view. Further more, we mcreasea TBP concentration and gave up the use of thiourea and the result wasn＇t improved. Through centrifugation protein precipitations appeared at room temperature. The short half-life of TBP might result in the poor effect in 2-DE. This study offers technical support for sample preparation and the proteomics research of Qinchuan cattle muscle tissue.

关 键 词：秦川牛 双向电泳 还原剂 二硫苏糖醇(DTT) 三丁基膦(TBP) 半衰期 

分 类 号：S828.3[农业科学—畜牧学]